Journal: Frontiers in Molecular Neuroscience

Article Title: Enhancement of Astroglial Aerobic Glycolysis by Extracellular Lactate-Mediated Increase in cAMP

doi: 10.3389/fnmol.2018.00148

Figure Lengend Snippet: Inhibition of AC reduces the L -lactate- and 3Cl-5OH-BA-induced increase in [cAMP] i and [lactate] i (A,B , panels i) Mean time-courses of FRET ratio signal changes normalized to the maximum signal change for (A,i) Epac1-camps upon addition of 20 mM L -lactate and (B,i) Laconic upon addition of 0.5 mM 3Cl-5OH-BA (black lines) in the absence (white circles) and presence (black circles) of 100 μM DDA, an inhibitor of AC. Each data point represents mean ± s.e.m. ( A,B , panels ii) Mean relative changes in FRET ratio (Rel. ΔFRET ratio) upon L -lactate (A) and (B) 3Cl-5OH-BA stimulation in the absence and presence of DDA. Relative ΔFRET values (%) were calculated by dividing individual ΔFRET values with the average ΔFRET value upon L -lactate or 3Cl-5OH-BA stimulation. Note that the inhibition of AC by DDA causes ∼50 % reduction in L -lactate-induced increase in [cAMP] i in astrocytes and a ∼30–60% reduction in 3Cl-5OH-BA-induced increase in [lactate] i in astrocytes, 3T3-L1 and BT474 cells. In BT474 cells the application of 3Cl-5OH-BA initiated a transient reduction in [lactate] i that was diminished in the presence of DDA. Numbers adjacent to black bars represent number of cells. Data are in the format of the mean ± s.e.m ( ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001). Data for every set of experiment was acquired from at least two different animals.

Article Snippet: BT474 cancer cells (BT-474 Clone 5; ATCC ® CRL-3247 TM ; ATCC-LGC Standards) were grown in Hybri-Care medium (ATCC ® 46-X TM ; ATCC-LGC Standards) supplemented with 1.5 g/L sodium bicarbonate and 10% fetal bovine serum.

Techniques: Inhibition

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A – D ) Carfilzomib and bortezomib induce cell death in a concentration-dependent manner. (A) and (C) Equal numbers of BT474 or MDA-MB-361 cells were seeded on 12-well culture plates and treated with the indicated carfilzomib or bortezomib concentrations. After 10 days cells were fixed and stained. (B) and (D) BT474 and MDA-MB-361 cells were cultured in the presence of the indicated carfilzomib and bortezomib concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Concentration Assay, Staining, Cell Culture, Western Blot

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) BT474 and ( B ) MDA-MB-361 cells were cultured in the presence of the indicated carfilzomib (right panels) and bortezomib (left panels) concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Cell Culture, Western Blot

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) Carfilzomib and bortezomib induce cell death in a time-dependent manner. BT474 cells were cultured in the presence or absence of the indicated carfilzomib or bortezomib concentrations. The percentage of SubG1 cells was evaluated at different time points using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented. ( B ) BT474 cells were cultured in the presence of 250 nM carfilzomib or 25 nM bortezomib. Cells were harvested at the indicated time points. Western blots of protein lysates were probed with the indicated antibodies. ( C ) Schematic model of a possible molecular network regulated by both proteasome inhibitors, based on the results of the Western blots and the literature. Continued activation of HER2 has a causal role in tumorigenesis and leads to tumor progression [ , ]. Activation of HER2 triggers autophosphorylation of specific tyrosine residues within its cytoplasmic domain, consequently activating different intracellular signaling pathways such as Ras-MAPK and the PI3K/Akt axis which determine cell growth, cell survival and therapy resistance to endocrine therapies. Different protein tyrosine phosphatases regulate phosphorylation of the HER2 signaling domain and are therefore important key regulators of HER2 activity [ , ]. The PEST-type protein-tyrosine phosphatase BDP1 inhibits ligand-induced activation of HER2 [ , ]. PEST sequences cause accelerated degradation by the proteasome/ubiquitin system , and PIs therefore increase the amount of BDP1 through blocking its proteasomal destruction. This in turn leads to accelarated dephosporylation and thereby inactivation of HER2 and its downstream target signaling pathways. Aberrant signaling through HER2 and other members of the HER family mediates endocrine resistance in ER+ positive breast cancer. On the other hand ERα co-expression with HER2 attenuates the efficiency of anti-HER2-targeted therapies. These findings indicate that HER2 and ERα act in concert to allow breast cancer cells to escape from both anti-ERα and anti-HER2-targeted therapies. Besides inhibiting HER2, PIs also suppress ERα protein expression.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Cell Culture, Staining, Flow Cytometry, Western Blot, Activation Assay, Protein-Protein interactions, Phospho-proteomics, Activity Assay, Ubiquitin Proteomics, Blocking Assay, Expressing

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) Stable knockdown of BDP1 within BT474 cells was performed by using lentiviral transfer of non-targeted or ten different targeted shRNAs against BDP1. After transduction reduced expression of BDP1 in ten BT474 BDP1 k.o. cells was evidenced by qPCR (left panel). The two shRNAs (k.o.2 and k.o.7) with the highest potential to decrease BDP1 expression in BT474 cells at passage 2 of puromycin selection were used to quantify the degree of knockdown (right panel). ( B ) BT474 cells either transduced with non-targeted or BT474 k.o.2 and k.o.7 from passage 2 of puromycin selection were cultured in the presence or absence of carfilzomib for 28 hours. Western blots of protein lysates were probed with the indicated antibodies. ( C ) Equal numbers of BDP1 k.o.2, BDP1 k.o.7 and negative control scramble BT474 cells were seeded on 12-well culture plates and treated with the indicated carfilzomib concentrations (right panels). After 9 days cells were fixed and stained. ( D ) Macroscopic photos are shown of the fixed cell colonies for BT474 cells either untreated or treated with the indicated carfilzomib concentration. Bar 100 μm.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Knockdown, Transduction, Expressing, Selection, Cell Culture, Western Blot, Negative Control, Staining, Concentration Assay

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) Equal numbers of BT474 cells were seeded on 12-well culture plates and treated with the indicated lapatinib concentrations. After 10 days cells were fixed and stained (left panel). BT474 cells were cultured in the presence of the indicated lapatinib concentrations for 32 hours. Western blots of protein lysates were probed with the indicated antibodies (right panel). ( B ) BT474 cells were cultured in the absence or in the presence of the indicated concentrations of lapatinib, bortezomib or both drugs together (left panel) or BT474 cells were cultured in the absence or presence of the indicated concentrations of lapatinib, carfilzomib or in combination (right panel). After 30 hours cells were harvested. Western blots of the protein lysates were probed with the indicated antibodies. ( C ) Equal numbers of BT474 cells were seeded on 6-well culture plates and cultured either with or without the indicated lapatinib, bortezomib or carfilzomib concentrations. After 12 days cells were fixed. Macroscopic photos are shown of the fixed cell colonies for BT474 cells treated with lapatinib, bortezomib and lapatinib plus bortezomib (upper panel) and lapatinib, carfilzomib and lapatinib plus carfilzomib (lower panel). Bar 100 μm. ( D ) To determine the induction of cell death relative to the applied lapatinib and or bortezomib/carfilzomib concentrations equal numbers of BT474 cells were seeded on 12-well culture plates and cultured in the absence or in the presence of the indicated drug concentrations for 5 days. The percentage of SubG1 cells was evaluated using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Staining, Cell Culture, Western Blot, Flow Cytometry

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) BT474 cells were cultured in the absence or presence of lapatinib concentrations > 75 nM for 9 weeks and 15 passages. Untreated parental (BT474 p.) and BT474 cells which survived and continued to grow in the presence of lapatinib (BT474 LR.) were harvested. Protein lysates were assayed by immunoblotting for the level of ERα expression. β-actin served as loading control (left upper panel). Equal amounts (5 × 10 3 ) of BT474 p. and BT474 LR. cells were seeded per well on 6-well culture plates and cultured in the presence of 100 nM lapatinib. After 12 days cells were either fixed and stained (left lower panel) or cells were harvested after 5 days to determine induction of cell death. The percentage of SubG1 cells was evaluated using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented (right panel). P -values < 0.05 are indicated by asterisks. ( B ) BT474 LR. cells were cultured in presence of the indicated lapatinib, bortezomib or carfilzomib concentrations. Western blots of protein lysates were probed with the indicated antibodies. ( C ) Equal amounts of BT474 p. and BT474 LR. cells were seeded on 6-well culture plates and cultured either with or without the indicated concentrations of lapatinib, bortezomib or carfilzomib, respectively. For colony forming assay the cells were fixed and stained after 12 days cells (upper panel). Macroscopic photos of the fixed cell colonies are shown in the middle panel. Bar 100 mm (middle panel). To determine the induction of cell death cells were harvested after 5 days. The percentage of SubG1 cells was evaluated using propidium iodide staining and flow cytometry. Mean values ± s.d. of four independent experiments are presented (lower panel). P -values < 0.05 are indicated by asterisks.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Cell Culture, Western Blot, Expressing, Control, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Proteasome inhibitors prevent bi-directional HER2/estrogen-receptor cross-talk leading to cell death in endocrine and lapatinib-resistant HER2+/ER+ breast cancer cells

doi: 10.18632/oncotarget.20261

Figure Lengend Snippet: ( A ) BT474 cells were cultured in the absence or in the presence of the indicated concentrations and combinations of lapatinib, fulvestrant, carfilzomib and bortezomib. Where fulvestrant was used, it was administrated 4 hours before treatment with other drugs. After 30 hours cells were harvested. Western blots of the protein lysates were probed with the indicated antibodies. ( B ) To determine the induction of cell death in response to the indicated drug combinations equal numbers of BT474 cells were seeded on 12-well culture plates and cultured in the absence or in the presence of the indicated drug concentrations. The percentage of SubG1 cells was evaluated at different time points using propidium iodide staining and flow cytometry. Mean values ± s.d. of three independent experiments are presented. ( C ) Equal numbers of BT474 cells were seeded on 6-well culture plates and cultured either with or without the indicated drug combinations. After 8 days cells were fixed. Macroscopic photos are shown of the fixed cell colonies for BT474 cells either (a) untreated or treated with (b) lapatinib [125 nM] plus fulvestrant [1 μM], (c) bortezomib [30 nM], (d) bortezomib [30 nM] plus lapatinib [125 nM] plus fulvestrant [1 μM] or (e) carfilzomib [125 nM] and (f) carfilzomib [125 nM] plus lapatinib [125 nM] plus fulvestrant [1 μM]. Bar 100 μm.

Article Snippet: MCF7 and T47D cells were purchased from ATCC, BT474 cells were purchased from CLS Cell Line Services GmbH (Heidelberg, Germany) and MDA-MB-361 cells were purchased from Sigma Aldrich (Taufkirchen, Germany).

Techniques: Cell Culture, Western Blot, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Flow cytometric analysis of apoptosis induction in breast cancer cells treated with 20 μg/ml of immobilized and solution-phase rfhSP-D for 24 h.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques:

Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Apoptosis induction in BT20, BT474, and SKBR3 cell lines following HA challenge in the presence and absence of rfhSP-D (A) . The data were expressed as the mean of three independent experiments ( n = 3). A significant difference was seen among treated and untreated samples, as made evident by the shift in the fluorescence intensity. Staurosporine (1 μM/ml) was used as a positive control. Proliferative effects of rfhSP-D treatment on BT474 and SKBR3 breast cancer cell lines (B) . BT474 and SKBR3 cells were seeded in wells pre-coated with HA, HA + rfhSP-D, and rfhSP-D alone. The percentage of proliferative cells was evaluated by staining with mouse anti-human KI-67 antibody, and KI-67-stained cells were measured via flow cytometry. The data were generated from at least three independent experiments ( n = 3) and presented as mean ± SD (* p < 0.1, ** p < 0.01, and **** p < 0.0001). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells. The secretion levels of FL-SP-D were confirmed and analyzed via western blotting (C) . Culture medium collected from BT20, BT474, and SKBR3 cell lines was passed through a maltose Agarose column, and the eluted fractions were validated via western blotting; FL-SP-D was detected at ~43 kDa only for BT474. Both BT20 and SKBR3 cell lines did not secrete any FL-SP-D. Secreted FL-SP-D by BT474 was tested for its ability to induce apoptosis (D) . No effect of secreted FL-SP-D was seen in terms of cell viability and apoptosis induction.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques: Fluorescence, Positive Control, Staining, Flow Cytometry, Generated, Western Blot

Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Intracellular signaling to show phosphorylation of p53 in rfhSP-D-treated BT20 and SKBR3 cell lines (A) . Breast cancer cell lines were allowed to adhere to HA or HA-bound rfhSP-D, and the phosphorylation status of p53 was evaluated using total cell lysates with a PathScan Antibody Array Kit (Cell Signaling). Data were generated from at least three independent experiments and presented as mean ± SD. rfhSP-D treatment resulted in upregulation of p21 and p27 cell cycle inhibitors (B) . BT20, BT474, and SKBR3 (0.4 × 10 6 ) cells were seeded in a six-well-plate pre-coated with rfhSP-D and HA + rfhSP-D. The treated cells were lysed and pelleted down. The pelleted cells were subjected to RNA isolation, followed by cDNA synthesis and qPCR. The comparative quantification method was performed to calculate the efficiencies of each gene for each individual PCR and is based on the second differential maximum method or takeoff analysis. The takeoff results obtained with p21/p27 primers were normalized with the housekeeping gene TBP. qPCR assay was conducted in triplicates, and error bars represent ± SEM. Unpaired one-way ANOVA test was used to determine the significance; * p < 0.05, ** p < 0.01, and *** p < 0.001 ( n = 3). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques: Phospho-proteomics, Ab Array, Generated, Isolation, cDNA Synthesis